REVIEWThe autoimmune regulator: a key toward understanding themolecular pathogenesis of autoimmune polyendocrinopathycandidiasis-ectodermal dystrophy
Tanja Meriluoto,1,2 Maria Halonen,1,2 Markku Pelto-Huikko,3 Hannele Kangas,1 Juha Korhonen,1Meelis Kolmer,1,4 Ismo Ulmanen1 and Petra Eskelin1,2
1Department of Molecular Medicine, National Public Health Institute, Helsinki, 2Hospital for Children
and Adolescents, University of Helsinki, Helsinki, 3Departments of Developmental Biology and Pathology
Medical School, University of Tampere and Tampere University Hospital, Tampere(Received for publication on April 27, 2001)

Abstract. Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is an
autoimmune disease with autosomal recessive inheritance. APECED is characterized by the breakdown of tolerance to several organ-speciﬁc selfantigens. The symptoms of APECED fall into threemain categories: autoimmune polyendocrinopathies, chronic mucocutaneous candidiasis, and ectodermal dystrophies. The gene defective in APECED, AIRE, has been cloned and numerous mutationsin this gene have been found in patients with APECED. AIRE is predicted to encode a 545-amino-acidprotein containing structural domains characteristic for transcription regulators. The protein has beenshown to act as a transcriptional activator in vitro. The AIRE protein is mainly localized to the nucleus,
where it can be detected as speckles resembling nuclear bodies. In humans, the expression of AIRE has
been observed predominantly in immunologically relevant tissues, especially the thymus. Recently, we
have shown in the mouse that Aire is also expressed in various tissues and cell types outside the immune system. (Keio J Med 50 (4): 225–239, December 2001)
Key words: APECED, AIRE, APS I, autoimmunity, transcriptional regulation

Clinical Features of APECED
Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED; OMIM 240300), also
known as autoimmune polyglandular syndrome typeI (APS I), is a monogenic organ-speciﬁc autoimmune
disease affecting many tissues, mainly the endocrine
glands.1 Patients with APECED have a highly variable combination of destructive autoimmune reactions
toward different endocrine and nonendocrine organs,which may result in failure of for example the parathyroid glands, the adrenal cortex, the gonads, the pancreatic b-cells, the gastric parietal cells, and the thyroid
gland. The diagnosis is based on the occurrence of twoof the three major clinical manifestations, that is, primary atrophic hypoparathyroidism, primary adrenocortical failure (Addison’s disease), and chronic muco-

cutaneous candidiasis.2 Further, APECED may be
associated with gastrointestinal diseases such as autoimmune gastritis, malabsorption, and autoimmune hepatitis. The most characteristic ectodermal manifestations in APECED are dental enamel hypoplasia, pitted
nail dystrophy, and alopecia. Keratopathy, vitiligo, andcalciﬁcation of the tympanic membranes are also frequent. Table 1 summarizes the clinical ﬁndings in Finnish and Norwegian patients with APECED. The ﬁrst
symptoms usually appear in childhood, although theage of onset and the clinical phenotype vary widely,even within the same family. For all the patients withAPECED, life-long monitoring is needed for the appearance of new symptoms. A more detailed descriptionof the clinical aspects of APECED has been publishedelsewhere.2–4
Typical autoimmune manifestations of APECED are

Reprint requests to: Dr. Tanja Meriluoto, Biomedicum/ National Public Health Institute, Department of Molecular Medicine, PL 104, 00251
Helsinki, Finland, e-mail: tanja.meriluoto@ktl.ﬁ
4 Present Address: MediCel Ltd., Helsinki, Finland
225

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Meriluoto T, et al: APECED

Table 1 Frequency of Disease Components Among 68 Finnish and
20 Norwegian Patients with APECED

Table 2 Autoantigens Related to Clinical Manifestations in
APECED (Modiﬁed from Ekwall 2001)104

Disease component

Clinicalmanifestation

Frequency (%)
Finnish2
n ¼ 68

Norwegian4n ¼ 20

Endocrine components
79
72
60
4
12
13

85
80
31
10
0
0

100
77
13
72
52
18
12
35

85
40
25
40
10
10
5
10

Gonadal failure
Hypothyroidism
IDDM

Nonendocrine components
Autoimmune gastritis
Mucocutaneous candidiasis
Enamel hypoplasia
Vitiligo
Alopecia
Nail dystrophy
Malabsorption
Autoimmune hepatitis
Keratopathy* Calculated for post-pubertal individuals.

inﬁltration of lymphocytes in affected organs and thepresence of various circulating, tissue-speciﬁc autoantibodies (Ahonen P: Autoimmune polyendocrinopathycandidiasis-ectodermal dystrophy APECED. Doctoral
Thesis, Childrens’ Hospital, University of Helsinki,
Helsinki 1993). These antibodies may be responsible
for the destruction of the target tissues. Characteristic
target organs include the adrenal cortex and the gonads.
In patients with APECED, the autoantigens identiﬁed in these tissues are the cytochrome P450 enzymes
P450c17,5 P450c21,6 and P450scc,7,8 which are involvedin steroid synthesis.9 The appearance of the corresponding autoantibodies foreshadows the developmentof adrenal and ovarian failure.10 Other autoantigens
belonging to the group of cytochrome P450 enzymes
are CYP1A211,12 and CYP2A6.13 These antibodies areassociated with autoimmune hepatitis.13 In addition to
the autoantibodies targeted against the enzymes catalyzing steroid synthesis, another group of enzymatic
autoantigens is found in patients with APECED, including tryptophan (TPH),14 tyrosine (TH)15 and phelylalanine hydroxylase (PAH).16 These three enzymes
constitute a group of pteridin-dependent hydroxylases
functioning in the production of the neurotransmitters
serotonin and dopamine, together with aromatic Lamino acid decarboxylase (AADC).16 The TPH autoantibodies are associated with gastrointestinal dysfunction,14 whereas the presence of TH autoantibodies

Reference

Endocrine
Hypoparathyroidism
Addison’s disease

Hypoparathyroidism
Addison’s disease
Gonadal failure*
Hypothyroidism
IDDM
Autoimmune gastritis

Antibody against

Calcium sensing receptor
P450c21P450c17a
P450scc
P450scc
TPO
TG
ICA
GAD65
IA-2
HþKþ-ATPase

105
6
5
7
8
3
3
21
19, 20106
107

Transcription factors
SOX9 & SOX10TH
TPH
CYP1A2CYP2A6AADC

104

Nonendocrine
Vitiligo
Alopecia
Malabsorption
Autoimmune hepatitis

17
14
11, 12
13
12, 18

TPO ¼ Thyroid peroxidase, TG ¼ Thyroglobulin, ICA ¼ Islet cell
antibody, GAD ¼ Glutamic acid decarboxylase, IA-2 ¼ Tyrosine
phophatase IA-2, TH ¼ Tyrosine hydroxylase, TPH ¼ Tryptophanhydroxylase, CYP ¼ Cytochrome P450, AADC ¼ Aromatic L-aminoacid decarboxylase.

correlates with alopecia areata.17 So far, the link between
PAH and the clinical manifestations of APECED remains unknown. The appearance of insulin-dependent
diabetes mellitus (IDDM) is generally predicted wellby the antibodies against AADC,12,18 glutamic aciddecarboxylase (GAD65),19,20 and pancreatic cytoplasmic islet cell antibodies (ICA).21 However, althoughthese antibodies are often present in high titers inpatients with APECED, they seem to be of no value forpredicting IDDM in association with APECED. For
example, although more than 50% of the patients withAPECED have islet cell speciﬁc GAD65 autoantibodies, only 10–20% develop clinical IDDM.22 For areview of target autoantigens in APECED, see Eisenbarth (1999).23 The major autoantibodies related to theclinical ﬁndings in APECED are listed in Table 2.
In addition to APECED, only two monogenic autoimmune diseases, autoimmune lymphoproliferative syndrome (ALPS) types I and II, are known. ALPS type I
is caused by inactivating mutations in either the Fasgene or the Fas-ligand gene,24,25 and ALPS type II
results from mutations in the caspase 10 gene.26 Thus,
because of its monogenic etiology, APECED may be

Keio J Med 2001; 50 (4): 225–239

considered a unique model for organ-speciﬁc autoimmune diseases and, as such, could provide insights
into the pathogenesis of more common but geneticallycomplex autoimmune disorders.
Genetics of APECEDAPECED is inherited as an autosomal recessive
trait. Although it is reported worldwide, in most populations it appears to be a rare disease. In the genetically isolated populations of the Finns,2 the IranianJews,27 and the Sardinians28 the lifetime prevalenceof APECED is rather high (1 : 25,000, 1 : 9,000, and1 : 14,500, respectively). APECED is also relatively
common in Norway,4 Northern Italy,3 and Sweden(unpublished observations, 2001). APECED is one
component of the ‘‘Finnish disease heritage’’.29 The
expected homogeneity of the mutations in this group of
diseases offers a valuable advantage for mapping thecausative genes.30,31 By linkage analysis, using data of
Finnish families and a random mapping approach, weassigned the APECED locus to chromosome 21q22.3.32
By ancient recombinations observed in APECED haplotypes, the critical chromosomal region was restricted
to 350 kilobases. We were also able to demonstrate locus
homogeneity for APECED among patients from otherpopulations.33 Haplotype segregation analysis indicatedthat a spectrum of different mutations in the same
genomic region was responsible for the disease in all
the ethnic groups studied.
In the physical mapping of the gene, we utilized visual mapping techniques based on ﬂuorescence in situ
hybridization (FISH).34,35 To order and orientate thephysical clones reliably, we used the ﬁber FISH method.
The ﬁnal physical map was constructed by using the P1,
PAC, and BAC libraries as well as the chromosome 21speciﬁc cosmid library.36 In order to identify positional
candidates for APECED, we used direct cDNA selection,37 exon trapping,38 and large-scale genomic sequencing followed by computer-assisted analysis. A total of 87 kilobases of a continuous genomic sequence
proximal to the gene encoding liver-type phosphofructokinase (PFKL)39 was analyzed by gene prediction programs, such as GRAIL40 and Genie.41 Althoughseveral gene predictions were located in this genomic
sequence, one novel gene was recognized by the various
gene identiﬁcation programs. This proved to be thecausative gene of APECED.42,43
The gene that is defective in APECED comprises 14
exons spanning 11.9 kilobases of genomic DNA (Fig.
1). The intron-exon boundaries follow the GT-AG
rule.44 Analysis of the 3’ end of the gene implied thatthe last exon of the gene overlaps with the promoter
region of the PFKL gene transcribed from the same
strand.39 A putative promoter containing a TATA box,

227

a GC box, and a CpG island was identiﬁed immediately
upstream of the ﬁrst exon of the gene. The mutations
found in this gene in the Finnish patients with APECED
determined it to be the causative gene of APECED. The
cDNA corresponding to the gene was isolated from ahuman adult thymus cDNA library. This cDNA sequence exhibits a high GC content of 68.8% and contains an open reading frame (ORF).42
The same gene was independently identiﬁed as the
APECED gene and named the autoimmune regulator(AIRE) by Nagamine K, et al.43 Nagamine K, et al. also
reported multiple mRNA species resulting from alternative splicing. By means of Northern blot analysis, we
identiﬁed in several tissues the expression of a transcript of @2 kilobases, the highest mRNA levels being
found in the thymus, pancreas, and adrenal cortex,
whereas Nagamine K, et al. identiﬁed transcripts only inthe thymus, lymph node, fetal liver, and appendix.
The AIRE Protein
AIRE is predicted to encode a 545-amino-acid proline-rich protein. In vitro translation of the cDNA revealed a 58-kilodalton polypeptide, which is in conformity with the calculated molecular weight of 57.7
kilodaltons. The predicted secondary structure of theprotein consists mostly of coils, which is concordant
with its high proline content (11.7%). Analysis of the
primary amino acid sequence disclosed that the AIRE
protein contains a putative nuclear targeting signal between amino acids 113 and 133, implying that it has a
nuclear location. The AIRE protein also contains several structural domains characteristic for transcription
regulators (Fig. 1). The protein has two zinc ﬁngersof the plant homeodomain (PHD) type. These modular
domains are found in many nuclear proteins that have arole in the chromatin-mediated regulation of transcription.42,45 In addition, the AIRE protein harbors four
nuclear receptor-binding LXXLL domains and an inverted LXXLL domain, motifs that have been shown
to mediate the interactions of the nuclear proteins involved in transcriptional regulation.46–48 Further, theamino acid sequence of AIRE includes a putative
DNA-binding domain SAND,49 which has been identiﬁed in several nuclear proteins, including Sp100, Sp140/
LYSP100,50 IFN-induced nuclear phosphoproteins,51
DEAF-1-related proteins,52 suppressin,53 and the glucocorticoid modulatory element binding protein.54 Besides the zinc ﬁngers and the SAND domain, the highly
conserved N-terminal domain in the AIRE protein also
shares a signiﬁcant degree of homology with the homogeneously staining region (HSR) domain of the Sp100
and Sp140 proteins.49 The HSR domain, also named
the ASS domain,55 has been shown to be involved in
the homodimerization of several Sp100-related pro-

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Meriluoto T, et al: APECED

Fig. 1 Schematic presentation of the genomic structure of the AIRE gene, the AIRE protein, and the mutations described in AIRE. The
mutations marked with black symbols have been identiﬁed by us.

teins.50,56 Recently, the HSR domain has been reported
to mediate the homodimerization of the AIRE protein,
as well.57 A mouse homologue of AIRE (Aire) has beencloned and shows conservation of all the predictedfunctional domains of its human counterpart.55,58,59
Subcellular Localization and Function of the AIRE
Protein
The correct function of a protein depends on its
proper subcellular location. We and others have shownby both in vitro and ex vivo systems that the AIRE/Aireprotein is mainly localized to the cell nucleus.60–63 Immunoﬂuorescence staining demonstrated that, in transiently transfected COS-1, CV-1, HeLa, and NIH3T3
cells, the bulk of the AIRE/Aire protein is located innuclear speckles resembling nuclear bodies (Fig. 2A).
Interestingly, in COS-1 cells the number and size of thenuclear dots varies from cell to cell, which may reﬂect
modulation of the nuclear structures, for example, during the cell cycle. Further, by immunohistochemical
staining, AIRE was detected in the nuclei of cells frommultiple immunologically relevant human tissues, suchas the thymus, spleen, and lymph nodes. In these tissues,

the nuclear staining was observed in restricted subpopulations of cells.60
Recently, the AIRE protein has been reported tointeract with the CREB-binding protein (CBP) in
vitro,57 which suggests that, under certain conditions,
AIRE might occur in the same nuclear structures as the
CBP. The CBP is highly enriched in the PML (promyelotic leukemia) nuclear bodies,64–66 and consequently the AIRE protein might associate with these
nuclear structures, also. This assumption is further supported by the ﬁnding that one of the main componentsof the PML body, the Sp100 protein,67 shares HSR and
SAND domain homology with AIRE, and it has beensuggested that Sp100 and AIRE arose from a common
ancestor.49 Furthermore, it has been shown that the
HSR domain of Sp100 is able to interact with PML
bodies.68 However, double immunoﬂuorescence analysis performed in transiently transfected COS-1 cells
overexpressing the AIRE protein failed to demonstratesigniﬁcant co-localization of AIRE and the PML or
Sp100 proteins.60,62 The PML bodies are dynamic
structures with a heterogeneous constitution that depends on the particular phase of the cell cycle.69 Thus,
further studies are needed to assess the possible co-Keio J Med 2001; 50 (4): 225–239

229

Fig. 2 Subcellular localization of the mouse and human Aire/AIRE proteins in NIH3T3 and BHK cells, respectively. NIH cells were transiently transfected with wild-type mouse Aire cDNA (A, B). BHK cells were infected with recombinant Semliki Forest virus encoding the wildtype human AIRE (C). (A) Mouse Aire showed a distinct speckled distribution in the nucleus. (B) Some cytoplasmic ﬁlamentous staining ofcells expressing mouse Aire was observed. (C) Human AIRE was detected both as small dots in the nucleus, excluding the nucleoli, and aslarger granules in the cytoplasm.

localization of the AIRE protein and the PML bodies,preferably in cell lines expressing AIRE endogenously.
PML bodies seem to have various functions. They havebeen suggested as being involved in chromatin-mediated regulation of transcription,56 as well as in theregulation of cellular growth and transformation,70 tumor suppression71 and apoptosis.72–74 Recently, PML
bodies have also been associated with proteasomemediated protein degradation.75–77 Whether AIRE is
involved in any of the indicated functions of the PML
bodies, in addition to its presumptive function as atranscription regulator, remains to be shown.57,78
In transiently transfected tissue culture cells, bothhuman and mouse AIRE/Aire also show a cytoplasmic
vimentin or microtubule-like distribution (Fig. 2B).
Furthermore, some cytoplasmic aggregates may occasionally be observed and the proportion of these different distribution types varies with the cell line (Fig.
2C)60,63 (unpublished observations, 2001). In the transient overexpression systems, cytoplasmic aggregates of
the AIRE protein could be the result of saturated nuclear transportation mechanisms.60 Alternatively, theymight be a consequence of imbalance between appropriate cellular targets and excessive expression of theprotein. In the latter case, the nuclear vs. cytoplasmic
location of AIRE could depend on the availability of aprotein complex partner, which is needed for efﬁcienttranslocation to the nucleus.63 In immunohistochemical
staining of mouse tissue sections, some cytoplasmic localization of Aire was detected in certain cell populations, as well.63 This suggests that the cytoplasmic
distribution of AIRE may be biologically relevant.
Interestingly, the PML and the Sp100 proteins havebeen shown to be located in the nucleus as well as in thecytoplasm.79
Both the speckled nuclear localization and the structural motifs of the AIRE protein suggest that it has arole in the regulation of transcription. To study this hypothesis, we employed a eukaryotic one-hybrid expression assay, the principles of which are shown in Fig. 3A.
We were able to show that, in this transactivation assay,
the wild-type AIRE, when fused with a yeast DNAbinding domain, strongly activates the transcription of
the luciferase reporter gene in vitro. Our results provided, for the ﬁrst time, experimental evidence that the
AIRE protein is a powerful transcriptional activator.78
Concordantly with our results, Pitkänen J, et al. havealso recently shown that the AIRE protein has transcriptional transactivation activity.57
Tissue Expression of the AIRE/Aire Gene
Unraveling the tissue distribution of AIRE is essential for understanding the biological role of the protein.
In humans, AIRE protein expression is found in thethymus, where the expression levels are high, the lymph
nodes,60,61 the spleen, and the peripheral blood cells.60
The mRNA expression pattern is similar, and the appendix and the fetal liver also show expression.43,61
Interestingly, various studies on the expression of themouse Aire gene suggest a wider tissue expression pattern than that detected in the human tissue studies.
Aire mRNA has been detected by the RT-PCR technique in the thymus and spleen, and also in the peripheral blood cells, in the ovary, lungs, heart, and adrenal
and thyroid glands, the skeletal muscle, kidney, testis,

230

Fig. 3 Mammalian one-hybrid assay. (A) The AIRE cDNA fused inframe with the Gal4 DNA-binding domain (DBD) was co-transfected
into COS-1 cells with the luciferase reporter plasmid. In order to observe the effects of the AIRE-Gal4-DBD on the transcription activityof the reporter gene, luciferase activity was measured. (B) Transcription activity of the wild-type and mutant AIRE proteins in mammalian one-hybrid assay. The wild-type acted as a powerful transcription
activator. The activity of the wild-type AIRE is taken as 100%. The
Iranian Jewish mutant (254A>G) AIRE protein, with a disrupted
HSR domain, was a strong activator of the luciferase reporter gene.
Neither the AIRE protein carrying the major Finnish mutation(769C>T), without either PHD ﬁnger domain, nor the mutant AIRE
protein (1193delC), which lacks the second PHD ﬁnger, showed any
transactivation activity. The mutant AIRE (932G>A) with the ﬁrst
PHD ﬁnger disrupted had 30% transactivation activity.

lymph nodes, brain, and fetal liver.58,63,80,81 The expression of Aire has been observed in the mouse embryonic 17d kidney cDNA library.63 At the cellularlevel, in situ hybridization showed expression not onlyin the thymus, lymph node, bone marrow, and spleen,
but also in speciﬁc cells in the brain and the ovary.63
Aire protein expression has been detected by immunohistochemistry in several immunological and nonimmunological tissues. These results are summarized inTable 3 and Fig. 4. Our results on the expression of theAire protein disagree with those of an earlier study
in which Aire protein expression could not be observed

Meriluoto T, et al: APECED

by immunohistochemistry in the skeletal muscle, testis,ovary, kidney, adrenal gland, lung, lymph node, spleen
or liver.80 The Aire gene expression in different tissues
is restricted to certain subpopulations of cells ranging
from epithelial cells to neurons and glial cells. Consequently, in many of the tissues, this results in a low
overall expression level which, in turn, sets limits to thedetection capacity of the different methods used. The
conservation of the tissue antigenicity, the afﬁnity andspeciﬁcity of the antibody, and the quality of the probe
for in situ hybridization are the variables that mostprobably play an important role in explaining the contradictory results concerning the expression patternobtained by different research groups.
Further, the information provided by exponentiallygrowing public domain databases, such as dbEST(http://www.ncbi.nlm.nih.gov/dbEST/) and NCBI’s
project called ‘‘SAGE tag to gene mapping’’ (http://
www.ncbi.nlm.nih.gov/SAGE/) also indicates the expression of AIRE/Aire outside the immune system. To
date, more than three million human ESTs (expressedsequence tags) have been sequenced (dbEST database,release 033001, March 30, 2001). The only AIRE-speciﬁc EST identiﬁed so far originates from the germ celltumor cDNA library. Interestingly, out of two million
sequenced mouse ESTs, nine Aire-speciﬁc ESTs, fromthe thymus, mammary gland, and embryonic stem cell
cDNA libraries, have been characterized. Analyses ofhuman SAGE (Serial Analysis of Gene Expression)
libraries have yielded a total of almost four million
sequenced tags. Of these, 22 tags from various tissuesources are ascribed to the AIRE sequence. SAGE
data can be accessed through the human AIRE Unigene cluster Hs.129829 at http://www.ncbi.nlm.nih.gov/
UniGene/. The presence of the Aire protein outside theimmune system suggests that it may have several different biological functions.
Expression of the AIRE/Aire gene in the thymus has
been studied intensively. In both humans and themouse, the expression of the AIRE/Aire protein in thethymus is restricted to the medulla. Similarly, in themouse, the mRNA was found mainly in the thymus
medulla. At the cellular level, AIRE/Aire expression in
the adult is observed in the thymic corpuscles, the reticular epithelial cells, and in a subpopulation of the
medullary thymocytes.60,63,80,82 More speciﬁcally, a
subpopulation of the corticomedullary and medullary29þ epithelial cells express Aire in the adult tissue.
These cells are thought to function in the induction oftolerance through positive and negative selection.83,84
The development of the fetal thymic organ culture(FTOC) has enabled detailed studies of the roles of thedifferent stromal cell subsets in early thymopoiesis.
According to Zuklys S, et al.82 Aire mRNA is expressed only relatively late in ontogeny, after E14.

Keio J Med 2001; 50 (4): 225–239
Table 3231

The Expression of the Aire Protein in Different Mouse Tissues Detected by Immunohistochemistry

Organ

Cell type

Immune system
ThymusSpleenLymph nodes
Bone marrow
PBL

MedullaRed pulp
MedullaReticular epithelial, thymic corpuscle cells, medullary thymocytes
Tissue macrophages, lymphocytes, reticular cells
Lymphocytes, reticular cells
Megakaryocytes, lymphoblasts, myeloblasts
Lymphocytes, polymorphonuclear leukocytes, monocytes

Proximal and distal convoluted
tubules
Glomeruli
Kidney pelvis
Myometrium

Epithelial cells

Urinary tract
KidneyBladder

Podocytes
Transitional epithelium
Smooth muscle (weak)Transitional epithelium

Genital organs
TestisEpididymis
Seminal vesicle
Prostate
Ovary
UterusSeminiferous tubules
Interstitial cells

Follicles
MucosaSecretory glands
Myometrium

Pachytene spermatocytes, round spermatids, peritubular cells, Sertoli cells – few
Leydig cells
Epithelial cells
Epithelial cells
Epithelial cells
Granulosa cells, oocytes
Epithelial cells
Epithelial cells
Smooth muscle

Alimentary tract
Salivary glands
StomachSmall intestine and colon
Liver
Pancreas

Acini
Secretory ducts
MucosaMucosaSecretory cells
Epithelial cells
Mucosal epithelial, mucous secreting neck cells, parietal cells of gastric gland
Epithelial cells
Hepatocytes, Küppfer cells
Exo- and endocrine cells, Islets of Langerhans (most of the cells)

BronchiAlveolar sacks
Cartilage

Epithelial cells
Type-I epithelial cells, type-II alveolar cells, macrophages
Undifferentiated perichondrial cells, differentiating chondroblasts

Respiratory tract
Lung
Trachea
Endocrine organs
Adrenal gland

Thyroid gland
Pituitary gland

Zona glomerulosa
MedullaZona fasciculata and reticulata
Follicles
Anterior and intermediate lobe

Chromafﬁn cells
Epithelial cells, parafollicular cells

Nervous system
Brain

Cerebral cortex
Hippocampus
Amygdala
Thalamic nuclei
Hypothalamus
Cerebellar cortex
Spinal cord
Dorsal root ganglia
Various brain stem nuclei

Neurons
Glial cells

Eye
RetinaPBL: peripheral blood lymphocyte.

Ganglion cells
Bipolar neurons

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Meriluoto T, et al: APECED

Zuklys S, et al. assume that the triple negative (TN)thymocytes at stage II/III activate the expression of Airein the thymic epithelial cells (TEC). Cross-talk between
the TN thymocytes (stages I–IV) and TEC is required
for the development of the thymic stromal architecture.
The TN II/III thymocytes, in particular, are essential
for the formation of the proper three-dimensional cortical and medullary microenviroment needed for normal development of the T cells.85–87 This suggests that,as Aire gene expression is dependent on activation by
the TN II/III thymocytes, it is also dependent on a correct thymic stromal organization. It has been suggested
that the development of autoimmunity could be related
to an improperly organized thymic medulla (for a review, see Naquet P, et al.).88 An interesting ﬁnding isthat RelB-deﬁcient mice, in which the thymic architecture is irregular, and in which the activated major histocompatibility complex medullary epithelial cells areabsent,89,90 also lack Aire expression.80,82 These micealso have an increased number of autoreactive T cells in
the peripheral blood, suggesting that, although the thymocytes mature normally to single positive T cells, thenegative selection in the thymus may be impaired. The
absence of Aire expression may be explained by thelack of 29þ cells in the thymus of the RelB knockout
mice. So far, there is no experimental evidence fromwhich to conclude whether or not RelB, which is amember of the NF-kB family of transcription factors,91
directly regulates Aire gene expression.
Mutations in the AIRE Gene
So far, in patients with APECED, we and others
have described 34 different mutations.28,42,43,78,92–99
The mutations are spread throughout the coding regionof AIRE and consist of single nucleotide substitutions,
small insertions, and deletions. In addition, mutations
affecting splicing consensus sequences have been characterized (Fig. 1).95,96 We have recently systematicallyscreened the complete coding region and the intronexon boundaries of the AIRE gene of a series of 132
patients with APECED from different ethnic backgrounds.4,78,92 In these patients, we have characterized
16 different mutations. In about 5% of the patientsanalyzed, we have not identiﬁed any mutations. In addition, in another 5% of the patients we have foundonly one mutant allele of the AIRE gene. Since our sequence analyses did not cover the promoter region andthe introns, it is probable that the mutations are locatedin these sequences.
The Common Finnish and Sardinian Mutations
In the isolated populations in which enrichment of
APECED has occurred, a clear founder effect is ob-

served. In each of these populations, one common mutation has been detected in the majority of the disease
alleles. The common Finnish mutation, a C-to-T transition at the nucleotide position 769 in exon six, changes
an arginine into a premature STOP codon, and is predicted to lead to a truncated 256-residue protein lacking
both the PHD ﬁngers (R257X). By means of Western
blotting, the cDNA carrying the common Finnish mutation was shown to produce a truncated protein of 28
kilodaltons. Despite the intact nuclear targeting signal
this mutated AIRE protein was retained predominantlyin the cytoplasm, where it produced a ﬁlamentous
staining pattern (Fig. 5). In the one-hybrid expression
assay, the common Finnish mutation had totally abolished the transactivation capacity of AIRE (Fig. 3B).78
The R257X mutation has been shown to be the mostcommon mutation worldwide. We have observed thismutation in 90% of the Finnish and 30% of the nonFinnish APECED chromosomes (unpublished observations, 2001).42,78 It has also been reported to bepresent in the predominant Northern Italian APECED
allele.94 Both in Great Britain and North America, theR257X mutation is the second most common APECED
mutation.93,95,96 Haplotype segregation analyses provide strong evidence that in different populations theR257X mutation has independent origins.78,94 This is
not surprising, since recurring C-to-T transitions in CpG
dinucleotides, and particularly those in arginine codons,
are commonly described events.100
The common Sardinian APECED mutation, R139X,
is another example of a C-to-T transition occurring in a
CpG dinucleotide in an arginine residue.28 This nonsense mutation in exon three accounts for 90% of themutant alleles in the Sardinian patients with APECED.
However, the mutation seems to be unique to the Sardinians, and haplotype analyses in this population imply
that the APECED chromosomes carrying the R139X
mutation are all inherited from a single common ancestor. Thus, this mutation is unlikely to be a recurring
one. The R139X mutation was found in 1.7% of theSardinian control samples, indicating that, in this population the carrier frequency of APECED is relatively
high.
The Common Iranian Mutation
In the Iranian Jewish population, the lifetime prevalence of APECED has been estimated to be the highest
in the world (1 : 6 000–1 : 9 000).27 All the patients inthis population are homozygous for a single nucleotide
substitution, 254A>G, in exon two, resulting in a changeof the tyrosine into a cysteine, at position 85 in the Cterminal part of the HSR domain. This mutation has
not been detected in patients with any other ethnic
background or in the European control panel. One of

Keio J Med 2001; 50 (4): 225–239

233

Fig. 4 In situ hybridization and immunohistochemical staining of mouse Aire. (A) X-ray ﬁlm autoradiographs showing very strong signal for
AIRE mRNA in the medulla of thymus (arrows). The surrounding cortex shows the control level of the signal. (B) Dipped section showing
strongly labeled cells (arrows) in the medulla of the thymus. (C) Section showing grains in large epithelial cells (arrows) in the medulla of thethymus. Surrounding medullary thymocytes (arrowheads) lack the signal. (D) A small group of thymocytes (arrows) show a moderate signal for
AIRE. (E) Immunocytochemical demonstration of AIRE immunoreactivity (IR) in epithelial cells (arrows) in the medulla of the thymus. (F)Moderate staining can be seen in the medullary thymocytes (arrows). (G) Scattered AIRE-IR cells (arrows) are present in the medulla of thelymph node. (H) In the smear of peripheral blood leukocytes, clear labeling is seen in monocytes (mc), neutrophils granulocytes (ngc), andlymphocytes (lc). The arrow points to a non-labeled lymphocyte. (I) In the liver, most of the hepatocyte nuclei are strongly labeled. A minorityof the hepatocytes are unstained (arrows). Küpffer cells are also labeled (arrowheads). (J) High magniﬁcation showing heterogeneous distribution of AIRE-IR in the hepatocyte nucleus (arrows). (K) In the cerebellum, prominent labeling is seen in the nuclei (arrows) of the Purkinjecells ( pc), while granular cell (gc) nuclei (arrowheads) show modest staining (mc ¼ molecular layer). (L) In the testis, prominent staining isseen in seminiferous tubules, in the nuclei of the pachytene spermatocytes (arrows), and the round spermatids (arrowheads). (M) The interstitial cells of the testis are also labeled (arrows). (N) The cells of the zona glomerulosa (zg) of the adrenal cortex show nuclear labeling(arrows), while the cells of the zona fasciculata (zf) exhibit only cytoplasmic staining. (O) In the thyroid gland, the cells of the follicles show
variable labeling (arrows) and the parafollicular cells are clearly AIRE-IR (arrowheads). (P) Most of the cells in the parathyroid gland arestrongly AIRE-IR. Figures A to O are reproduced from Halonen et al. (2001).64 Bar: A ¼ 1 mm; B ¼ 100 mm; C, D, H ¼ 10 mm; E, G, I, L–P ¼
30 mm; F ¼ 60 mm; J ¼ 4 mm.

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Meriluoto T, et al: APECED

Fig. 5 Subcellular distribution of the mutant AIRE proteins in COS-1 cells. COS-1 cells were transfected with mutant AIRE expression constructs. The mutant carrying the major Finnish mutation, 769C>T lacking both PHD ﬁngers, was seen mainly in the cytoplasm as ﬁlamentous
staining. Mutant AIRE with the major Iranian Jewish missense mutation, 254A>G, was detected only in the nucleus, showing partly speckled
and partly diffuse staining. Most of the protein with the ﬁrst PHD ﬁnger disrupted, (932G>A) was localized in the cytoplasm as small dots. In a
few cells, some nuclear staining, without any distinct structures, was observed. The 1193delC mutant lacking the second PHD ﬁnger was seen
only in the cytoplasm as large granules.

the 35 unrelated, healthy Iranian Jewish controls washeterozygous for the Y85C mutation, which is inagreement with the high prevalence of APECED in thispopulation.78
In the vast majority of the transiently transfected
COS-1 cells, the protein harboring the common Iranian
Jewish mutation was transported into the nucleus. In
addition to the punctuate staining pattern, closely resembling that seen with the wild-type, it also produced
some homogeneous nuclear labeling (Fig. 5) (unpublished observations, 2001). This kind of even staining of
the nucleus was not detected in cells expressing thewild-type AIRE, which implies that the Y85C mutationin the HSR domain interferes with the subnuclear targeting of AIRE or the ability of the protein to form
distinct nuclear structures. Somewhat surprisingly, thecommon Iranian Jewish mutation had no major effecton the transactivation properties of the mutant protein

in vitro (Fig. 3B).78 Dimerization of the AIRE protein
may be necessary for DNA binding or for binding acurrently unknown partner. Mutations in the HSR domain could compromise these interactions. The onehybrid transactivation assay may fail to detect theeffects of mutations in such speciﬁc mechanisms. Thus,
the pathological consequences of the Y85C mutation
remain to be further addressed.
The Common British and Norwegian Mutation
A fourth mutation, observed in a relatively large
number of patients with APECED, is a deletion of 13
base pairs at the nucleotide position 965–977 in exon
eight. This leads to a frame shift and is predicted to
produce a truncated protein of 372 amino acids. Thisdeletion is the most common mutation among the British and Northern American patients with APECED

Keio J Med 2001; 50 (4): 225–239(71% and 56% of the APECED-causing mutations, respectively).93,95,96 It is also present in the predominant
Norwegian APECED allele.4 Furthermore, this 13 basepair deletion has been detected in affected chromosomes of individuals of many different geoethnic origins,
including the Finns, the Sardinians and the Northern
Italians.28,42,94 Haplotype analyses indicate that thedeletion has been independently introduced into many
populations several times. The DNA sequence surrounding the nucleotide 965 may be exceptionallyprone to mutations by slipped-strand mispairing, since
it contains several repeats of the CCTG sequence.93,101
Other Mutations in AIRE
Most of the mutations in the coding region of AIRE
are nonsense mutations leading to a premature STOP
codon and consequently to a truncated gene product.
The effects of the null mutations are dual: the predominant consequence of the nonsense mutations is not thesynthesis of a truncated protein. Rather, the majority
of nonsense transcripts are recognized and efﬁciently
degraded by the cell via a pathway known as nonsensemediated mRNA decay.102 To verify this mechanism,
transcripts or polypeptides should be studied in appropriate samples from the patients, which is complicatedby the low expression levels of the AIRE gene in mosttissues.
Altogether, we and others have characterized ten
missense mutations in AIRE.43,78,93,97,99 These mutations are of particular interest, since they may reveal
functionally critical regions of the AIRE protein. A
cluster of seven missense mutations, including the common Iranian Jewish mutation, occurs in the aminoterminal HSR domain, which has been reported to act asa homodimerization domain.43,78,93,97 Recently, Pitkänen J, et al. have shown that two of these mutations,
L28P and K83E, severely compromise both the dimerization and the transactivation function of AIRE in
vitro.57 Furthermore, our recent ﬁndings indicate that
mutations in this domain abolish the normal subnuclear
organization of AIRE (unpublished observations, 2001).
The HSR domain might play a role in determining thenuclear distribution of the protein and its association
with speciﬁc structures in the nucleus.
Three different missense mutations are localized to
the region coding for the ﬁrst PHD ﬁnger.78,99 The
conformational changes caused by these mutations may
disrupt the structure of the zinc ﬁnger. One of thesemutations, a G-to-A substitution at the nucleotide 932
changes the ﬁrst conserved cysteine residue of the PHD1
(amino acid 311) into tyrosine, and might thus compromise the co-ordination of the zinc atom. This, in turn,is deleterious to the three-dimensional structure of thezinc ﬁnger. In transiently transfected COS-1 cells, the235protein with the C311Y mutation was detected mainlyin the cytoplasm, where it formed small granules. In@10% of the cells, some nuclear staining was also seen,
but no distinct structures were observed (Fig. 5).78
These ﬁndings are in agreement with our earlier results
for the common Finnish mutation and a mutant lacking
the second PHD ﬁnger (1193delC), which showed
exclusively cytoplasmic localization (Fig. 5). The results
suggest that the proper nuclear targeting of AIRE depends upon the intact structure of the PHD ﬁngers.
Interestingly, in the transactivation assay, the mutant
protein with a disrupted ﬁrst PHD ﬁnger but an intact
second PHD ﬁnger showed about one-third of theactivity of the wild-type protein (Fig. 3B). Since a small
proportion of the native mutant protein was observedin the nuclei of the transfected cells, this mutant polypeptide may not be totally inactive.78 Pitkänen J, et al.
have recently reported results that are concordant withthese ﬁndings. They showed that a non-patient mutation, C437P, occurring in the second PHD ﬁnger was
also deﬁcient in transcriptional activation and resulted
in 30% activation compared with the wild-type AIRE.57
These results suggest that both the PHD ﬁngers are
involved in the transactivation function of AIRE. The
mutant without the second PHD ﬁnger totally lacked
the transactivation capacity, which further emphasizes
the role of the zinc ﬁngers in transcription activation(Fig. 3B).78
Conclusions
APECED represents a unique autoimmune disease
in several respects. It is a monogenic disease inherited
as an autosomal recessive trait and maps outside the
HLA region. Thus, the cloning of the defective gene,
AIRE, offers excellent possibilities for studying themechanisms of autoimmunity. The exact role of AIREin regulating the immune responses remains obscure.
However, the clinical ﬁndings in APECED suggest that
this gene may have an essential role in the development
and maintenance of self-tolerance. It has been hypothesized that, in the APECED disease, the tolerance of the
T cells is impaired. The tolerance is ﬁrst induced centrally in the neonatal period by negative selection andlater maintained in the periphery by ignorance, anergy,
homeostatic control, and regulation by dedicated T
cells.103 The expression of AIRE in the thymus occurs
in epithelial cells that are connected with negative selection of thymocytes and therefore may indicate a role
for AIRE in the central induction of tolerance. However, the expression of Aire elsewhere in the body suggests that other mechanisms may also be involved.
The presence of Aire in the target tissues of the
autoimmune destruction in APECED, such as theadrenal cortex, parathyroid gland, ovary, testis, and236

Meriluoto T, et al: APECED

pancreas, is intriguing but must await more detailed
characterization of the Aire expression during the organogenesis in the future. It will also be crucial to study
the transcriptional regulation of Aire and the functionalsigniﬁcance of Aire expression in relation to the tissue
background.
Recently, we have generated a knockout mousemodel for APECED (unpublished observations, 2001).
Despite the well-established differences in the immunological pathways between rodents and humans, theknockout model will be extremely valuable for the detailed understanding of the tissue destruction observedin patients with APECED. In the future, it might also
be helpful for developing novel therapeutic strategies
for APECED and perhaps also for other organ-speciﬁc
autoimmune diseases.
The localization of AIRE, as well as its mouse counterpart Aire, inside the nucleus provides us with a clue
to its biological functions. We have shown in vitro that
AIRE acts as a powerful transactivator. In order tocarry out their biological functions, the proteins participating in the regulation of transcription interact withseveral other proteins. Identiﬁcation of these interacting partners necessary for the regulatory activity of
AIRE remains to be explored. This will be essential forunderstanding the biological role of the protein and fordeﬁning the mechanisms triggering the destruction of
the target tissues in APECED. Further, characterization of the genes regulated by AIRE may unravel important new elements of the human immune system,and therefore will also shed light on the pathogenesis of
autoimmune diseases in general.
The knowledge obtained from the identiﬁcation of
the AIRE gene and the characterization of the numerous
disease-causing mutations in it has led to clinical applications in the form of a direct DNA diagnosis for
APECED. Further, it has been suggested that sequence
alterations in the AIRE gene might contribute to thepathogenesis of other, more common autoimmune diseases such as IDDM and Addison’s disease. The several
polymorphisms found inside the coding region of thegene may provide tools for studying the involvement of
AIRE in other organ-speciﬁc autoimmune diseases.
Acknowledgements: This study has been supported ﬁnancially by the
Academy of Finland (grants 50012 and 47396), Ulla Hjelt Fond of
the Foundation for Pediatric Research, Maud Kuistila Foundation,
the Emil Aaltonen Foundation and the Medical Research Fund of
Tampere University Hospital. Support from the Helsinki Biomedical
Graduate School MD/PhD program is also acknowledged.

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